Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates

Nucleic Acids Res. 2007;35(18):6052-62. doi: 10.1093/nar/gkm587. Epub 2007 Aug 30.


We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5' to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3' hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 2-Aminopurine / chemistry*
  • DNA Primers / chemistry*
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / metabolism
  • Deoxyribonucleotides / chemistry
  • Deoxyribonucleotides / metabolism*
  • Fluorescence
  • Kinetics
  • Nucleic Acid Conformation
  • Phosphates / chemistry
  • Spectrometry, Fluorescence
  • Templates, Genetic
  • Viral Proteins / chemistry*
  • Viral Proteins / metabolism


  • DNA Primers
  • Deoxyribonucleotides
  • Phosphates
  • Viral Proteins
  • 2-Aminopurine
  • DNA-Directed DNA Polymerase
  • bacteriophage RB69 DNA polymerase